Skoda et al (1988 P.N.A.S. 85, 5240-5243) disclosed an RFLP-based assay which identifies only about 25% of poor metabolisers.
Cytochrome P450-dependent monooxygenases (P450s) are a supergene family of enzymes that catalyse the oxidation of lipophilic chemicals through the insertion of one atom of molecular oxygen into the substrate. They are involved in the metabolism of xenobiotic compounds, and in particular with the clearance of at least 25 drugs, including debrisoquine, sparteine, bufuralol and dextromethorphan. Other drugs whose metabolism is related to the debrisoquine oxidation polymorphism (as of June 1990) include (in the cardiovascular area) metoprolol, timolol, propranolol, perhexilene, N-propylamaline, propafenone, encainide, flecainide and mexiletine, (in the psychiatric area) amitriptyline, imipramine, desipramine, nortriptyline, clomipramine, thioridazine, perphenazine, amiflamine and tomoxitene and (in other areas) codeine, methoxyphenamine and phenformin and possibly also chlorpropamine, melatonin and MPTP. The P450 system is polymorphic in man, and genetic differences in the P450-mediated metabolism of a wide variety of drugs have been clearly demonstrated. The best example of this is the debrisoquine/sparteine polymorphism, (see Ref 1 for a review). Up to 10% of the Caucasian population exhibit the poor metaboliser (PM) phenotype. This is characterised by a significantly reduced ability to metabolise the prototype drug debrisoquine to 4-hydroxydebrisoquine, the metabolism being 10-200 times less than in extensive metabolisers (EMs). The PM phenotype is inherited as an autosomal recessive trait, and up to 54% of people are carriers of a mutant allele(s). The PM phenotype leads to impaired clearance of over twenty other commonly prescribed drugs and may result in serious adverse side effects upon their administration. Thus the ability to predict phenotype is an attractive possibility which would be useful in many clinical situations.
Recently the cytochrome P450 isozyme (P450 db1, also called P450 buf 1 or P450 DB) responsible for the metabolism of debrisoquine, sparteine and other compounds related to the polymorphism has been purified from human liver. Immunoquantitation of this protein correlates well with the levels of bufuralol-1'-hydroxylase activity in a series of human livers, bufuralol being a prototype substrate for the db1 isozyme. Furthermore no immuno-reactive db1 protein was detected in liver microsomes of PMs suggesting that the complete or almost complete absence of this protein leads to the PM phenotype. Recent work also provides evidence for the presence of allozymes of P450db1 with altered K.sub.m and V.sub.max probably due to amino acid substitutions. Antibodies against human P450db1 have been found in patients with autoimmune hepatitis type II but the relationship between the debrisoquine polymorphism and the appearance of these autoantibodies is not known.
Gonzalez and coworkers have isolated cDNA clones from libraries made from the livers of EMs and have shown that they encode active P450db1 by expression in COS-1 cells and measurement of bufuralol-1'-hydroxylase activity (2). Sequence analysis shows that P450 db1 belongs to a distinct P450 subfamily, P450IID (3). P450IID cDNA clones were also obtained from libraries made from the livers of PMs and in these cases they appeared to be derived from aberrantly or incompletely spliced mRNAs, and therefore would not be able to encode an active P450db1. Four variants were described: "a" which retains intron 5; "b" which retains intron 6; "b'" which has lost the 3' half of exon 6 in combination with the removal of intron 6; and another cDNA clone from a PM liver, variant "c", which appears to be normally spliced but has several base substitutions and was not characterised further. It was inferred from these studies that the defective mRNAs (cDNAs) were the products of mutant alleles of the P450db1 gene.
The gene encoding P450db1 (CYP2D1) has been located on chromosome 22 and Southern blot analysis shows that there is probably more than one gene/pseudogene within the CYP2D locus based on the amount of DNA hybridizing to the db1 cDNA probe. The CYP2D locus is highly polymorphic, and two alleles, detected using the restriction enzyme XbaI, have been associated with the PM phenotype (44 kb allele and 11.5 kb allele; 4). However, at the present time these restriction fragment length polymorphisms (RFLPS) are not informative in predicting phenotype as they do not identify all PM individuals (4).